Process for producing sporangia of bacillus popilliae

ABSTRACT

The present invention provides a process for producing sporangia of  Bacillus popilliae  containing spores and parasporal bodies in large numbers per unit volume of medium. In a process for producing sporangia of  Bacillus popilliae  containing spores and parasporal bodies by culturing  Bacillus popilliae  in a liquid medium containing an adsorbent, the liquid medium contains 0.1-0.7% by weight of proline.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for producing sporangia ofmicroorganisms belonging to Bacillus popilliae containing spores andparasporal bodies useful as a control agent of Scarabaeidae insects(“sporangia containing spores and parasporal bodies” may simply bereferred to as “sporangia”) by culturing Bacillus popilliae in liquidmedium.

2. Description of the Related Art

The larva of Scarabaeidae insects feed on a wide range of plant rootssuch as those of grasses, agricultural and horticultural crops andtrees, and are known to cause considerable damage. Since these larvalive underground, it is difficult obtain control effects by sprayingagricultural chemicals from the air, and it is difficult to identify thelocations where these larva are present. Therefore, it has beennecessary to spray large amounts of agricultural chemicals over a widerange to enable the chemicals to penetrate into the ground, and sincethere are concerns over detrimental effects on both the naturalenvironment and people, a more effective control method is desired.

Microorganisms belonging to Bacillus popilliae are known toparasitically cause milky disease in the larva of Scarabaeidae insects,and eventually cause their death. Consequently, attempts have long beenmade to use the sporangia of these microorganisms to controlScarabaeidae insects on which agricultural chemicals have little effect.

For example, an example of a production process is described in JapaneseUnexamined Patent Application, First Publication No. 2001-149066 inwhich a sporangia formation rate (ratio of number of sporangia to numberof microbial cells) of 4.8% is obtained by culturing Bacillus popilliaein solid medium containing 0.05-0.5% by weight of activated carbon.However, culturing methods using solid medium have the problem of lowproductivity.

Various studies have been conducted on culturing methods using liquidmedium in order to solve this problem of the aforementioned culturingmethod using solid medium. For example, Haynes, et al. reported anexample of attempting to culture Bacillus popilliae NRRL B-2390S inliquid medium containing 0.5% peptone, 1.5% yeast extract, 0.3%dipotassium hydrogenphosphate, 0.1% glucose and 1% activated carbon(Journal of Invertebrate Pathology, Vol. 22, p. 377-381, 1973). However,only a maximum of 2.06×10⁷ sporangia per 1 ml of liquid culture wereobtained, thus resulting in the problem the concentration of sporangiabeing too low to achieve higher productivity.

In addition, Haynes, et al. also reported that 3.1×10⁷ sporangia per 1ml of liquid culture were obtained by culturing mature cells of Bacilluspopilliae NRRL B-2309S in the late logarithmic increase stage in liquidmedium containing 0.5% peptone (tryptone), 1.5% yeast extract, 0.3%dipotassium hydrogenphosphate, 0.1% glucose and 1% activated carbon(Journal of Invertebrate Pathology, Vol. 19, p. 125-130, 1972). However,this culturing method has a long culturing time, taking roughly twoweeks.

Moreover, an example of having obtained 1×10⁹ sporangia per 1 ml ofliquid culture by culturing in liquid medium containing 1% solublestarch, 0.1% trehalose, 0.5% yeast extract, 0.3% dipotassiumhydrogenphosphate and 0.1% calcium carbonate is indicated in U.S. Pat.No. 4,824,671. However, there were no parasporal bodies present in thesporangia, and as a result, the rate of infection with milky diseasewhen sporangia were sprayed at the rate of 2.0×10¹² sporangia per 1 kgof soil and allowed to be orally ingested by larva of Scarabaeidaeinsects was 47.50% after the passage of 7 weeks, indicating weakinsecticidal effects on larva of Scarabaeidae insects even when comparedwith sporangia containing parasporal bodies formed within the bodies ofthe larva.

BRIEF SUMMARY OF THE INVENTION

The object of the present invention is to provide a process forproducing sporangia of microorganisms belonging to Bacillus popilliae ofwhich a large number are produced per unit volume of medium.

In order to solve the aforementioned problems, the present inventionprovides a process for producing sporangia of microorganisms belongingto Bacillus popilliae containing spores and parasporal bodies byculturing Bacillus popilliae in liquid medium containing an adsorbentand 0.1-0.7% by weight of proline.

According to the production process of the present invention, 5×10⁷sporangia or more of Bacillus popilliae containing spores and parasporalbodies can be produced per 1 ml of liquid culture and at a highsporangia formation rate of 6-50% by liquid culturing for about 5-10days. In addition, the number of sporangia produced per unit volume ofmedium can be further increased by adding pyruvic acid to the liquidmedium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing a sporangium of Bacillus popilliaecontaining a spore and a parasporal body.

FIG. 2 is a graph showing the relationship between the number ofsporangia containing spores and parasporal bodies produced versusproline concentration in liquid media in Examples 2-4 and ComparativeExamples 5-6.

FIG. 3 is a graph showing the growth inhibitory effects on Anomalacuprea in Biological Test Example 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to Bergey's Manual of Determinative Bacteriology, EighthEdition, the bacteriological properties of the microorganisms belongingto Bacillus popilliae used in the present invention consist ofmorphological properties including being Gram negative bacilli having alength of 1.3-5.2 μm and width of 0.5-0.8 μm, a growth temperature of20-35° C., and the sporangium 1 having a spore 3 and parasporal body 2inside as shown in the schematic drawing of FIG. 1. However, it has beenproposed, based on the theoretical opinions of Pettersson, et al. (Int.J. Syst. Bacteriol., Vol. 49, p. 531-540, 1999), that Bacillus popilliaeshould be reclassified as Paenibacillus popilliae. In addition, Rippere,et al. (Int. J. Syst. Bacteriol., Vol. 48, p. 395-402, 1998) andHarrison, et al. (J. Invertebr. Pathol., Vol. 76, p. 169-175, 2000) haveproposed that the milky disease pathogens of Bacillus popilliae andBacillus lentimorbus be classified at the DNA level since they cannot beclearly distinguished on the basis of only the presence or absence ofparasporal bodies and the presence or absence of growth in 2%salt-containing medium, which had been used to distinguish the twospecies in the past. Since their classification is not clearly definedat present, the microorganisms belonging to Bacillus popilliae in thepresent invention are to include microorganisms belonging toPaenibacillus popilliae and microorganisms belonging to Paenibacilluslentimorbus.

The liquid medium used in the present invention contains an adsorbentfor the purpose of removing substances that inhibit the growth ofBacillus popilliae. Examples of said adsorbent used for this purposeinclude activated carbon, adsorbent resin, allophosite and molecularsieve, and activated carbon is particularly preferable. Although theactivated carbon used for the adsorbent may be in the form of a powder,granules or a sheet and so forth, powdered activated carbon isparticularly preferable because it has the highest decomposition oradsorption ability per unit weight.

Adsorbent resin refers to a porous polymer that has adsorbentproperties, examples of which include crosslinked porous polymers moldedinto granules which function as a synthetic resin capable of efficientlyadsorbing growth inhibiting substances in aqueous solution due to amicroporous structure that extends inside the granules. Examples ofadsorbent resins include the aromatic synthetic resin adsorbentsmanufactured by Mitsubishi Chemical under the trade names of DiaionHP20, Diaion HP21, Sepabeads SP825, Sepabeads SP850, Sepabeads SP70 andSepabeads SP700, the substituted aromatic synthetic resin adsorbentmanufactured by Mitsubishi Chemical under the trade name of SepabeadsSP207, and the acrylic synthetic resin adsorbent also manufactured byMitsubishi Chemical under the trade name of Diaion HP2MG.

The content of adsorbent in the liquid medium used in the presentinvention is preferably 0.05-5% by weight of the liquid medium. Bymaking the content of adsorbent in the liquid medium 0.05% by weight ormore, adsorption and elimination effects on substances that inhibitmicroorganism growth tend to be adequately demonstrated, and by makingthe content of adsorbent in the liquid medium 5% by weight or less, theadsorption of nutrient sources required for growth of the microorganismstends to be minimized, which is preferable since a high level ofmicrobial growth promotional effects for Bacillus popilliae aredemonstrated within the aforementioned range.

The proline content in the liquid medium used in the present inventionis preferably 0.1-0.7% by weight, and particularly preferably 0.2-0.6%by weight, of the liquid medium. If the content of proline in the liquidmedium is less than 0.1% by weight or if it exceeds 0.7% by weight, inaddition to lowering microbial growth promotion effects, the number ofsporangia formed per unit volume of medium also decreases.

Medium should be used for the liquid medium used in the presentinvention that contains proline and adsorbent at the aforementionedcontent ratios in a known liquid medium used to culture Bacilluspopilliae.

Examples of components contained in such known liquid media includenitrogen sources, carbon sources and inorganic salts.

Examples of nitrogen sources include inorganic nitrogen sources normallyused for culturing microorganisms such as ammonia, nitric acid and saltsthereof, and organic nitrogen sources such as peptones, meat extract,fish extract, lactoalbumin hydrolyzates and yeast extract. Among these,peptones, lactoalbumin hydrolyzates and yeast extract are particularlypreferable. The content of nitrogen sources in the liquid medium used inthe present invention is preferably 0.001-5% by weight, and particularlypreferably 0.2-4% by weight.

Although various amino acids are contained in the aforementionednitrogen sources, and proline is included in these amino acids, theproline content is extremely low. In the production process of thepresent invention, proline is added separately to a known liquid mediumso that the ratio of proline to total amino acids in the liquid mediumis preferably 10-65% by weight, and particularly preferably 25-50% byweight.

The aforementioned total amino acids refer to 16 types of free aminoacids composed of alanine, arginine, aspartic acid, glutamic acid,glycine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, histidine, tyrosine and valine that areknown to be contained in nitrogen sources such as peptones and yeastextract ordinarily used as components of liquid media. The total amountof these 16 types of free amino acids is frequently used to roughlyindicate the total amount of free amino acids contained in peptones,yeast extract and so forth.

Examples of carbon sources that can be used as carbon sources in thepresent invention include sugars such as starches, trehalose andsucrose, and agricultural waste products such as waste honey, starchdegradation products and cheese whey. The content of these carbonsources is preferably 0.001-5% by weight of the liquid medium. However,although glucose is suited for growth of Bacillus popilliae, since ittends to inhibit the formation of sporangia containing spores andparasporal bodies, the concentration of glucose contained in the liquidmedium is preferably 0.01% by weight or less of the liquid medium.

Examples of inorganic salts include potassium chloride, sodium chloride,calcium chloride, sodium carbonate and phosphates normally used forculturing microorganisms, with phosphates being preferable, potassiumdihydrogenphosphate, dipotassium hydrogenphosphate and disodiumhydrogenphosphate being particularly preferable. The content ofinorganic salt is preferably 1% by weight or less of the liquid medium.

In addition to these components, other known additives may also be usedsuch as pH adjusters within a range that does not impair the effects ofthe present invention.

Among the components of the liquid medium described above, an example ofa liquid medium preferable for use in the production process of thepresent invention is shown in Table 1. TABLE 1 Liquid Medium Mediumcomponents (contents) or pH Example 1 Proline (0.1-0.7 wt %) Adsorbent(0.05-5 wt %) Peptone, yeast extract and lactoalbumin hydrolyzate(0.001-5 wt %) Trehalose (0.001-5 wt %) Water pH (6.5-8.5) Example 2Proline (0.1-0.7 wt %) Adsorbent (0.05-5 wt %) Peptone and yeast extract(0.001-5 wt %) Trehalose (0.001-5 wt %) Water pH (6.5-8.5)

In addition, pyruvic acid is also preferably added to the liquid mediumcontaining proline and adsorbent used in the present invention. Theaddition of pyruvic acid to the liquid medium further promotes growth ofBacillus popilliae, while also further increasing the number ofsporangia containing spores and parasporal bodies produced per unitvolume of medium. Furthermore, the pyruvic acid used in the presentinvention also includes physiologically acceptable salts thereof.Specific examples of physiologically acceptable salts of pyruvic acidinclude sodium pyruvate and potassium pyruvate.

The content of pyruvic acid in the liquid medium in the case of addingpyruvic acid to the liquid medium is preferably 0.01-0.5% by weight, andparticularly preferably 0.03-0.3% by weight, of the liquid medium. Bymaking the content of pyruvic acid in the liquid medium 0.01-0.5% byweight, a high level of growth promotional effects can be exhibited forBacillus popilliae, and the number of the aforementioned sporangia canbe increased per unit volume of medium.

The suitable culturing temperature for the growth of Bacillus popilliaeis 25-32° C. In addition, the pH of the liquid medium is preferably6.5-8.5, and particularly preferably 7-8. Examples of methods foradjusting the pH of the liquid medium include the addition of variousbuffers, the addition of routinely used acids such as hydrochloric acidor sulfuric acid, and the addition of routinely used bases such assodium hydroxide, potassium hydroxide or ammonia.

Liquid culturing may be carried out by any method, examples of whichinclude batch culturing, continuous culturing, semi-batch culturing andfeeding culturing. Although culturing time varies according to theculturing method, culture temperature, culture pH and number ofinoculated microorganisms, it is normally 5-10 days in the case of batchculturing.

Following completion of culturing, sporangia containing spores andparasporal bodies are recovered from the culture. This recovery shouldbe carried out by separating microbial cells containing said sporangiafrom the culture by centrifugation, filtration or other typicalseparation method. At this time, a washing procedure may be added usingwater or buffer as necessary.

According to the production process of the present invention, sporangiaof Bacillus popilliae can be produced at a sporangia formation rate of6-50%, and 5×10⁷ to 1×10⁹ of said sporangia can be produced per 1 ml ofliquid culture.

The sporangia of Bacillus popilliae containing spores and parasporalbodies obtained from the production process of the present invention areuseful as a control agent for Scarabaeidae insects by demonstratingcontrol effects such as insecticidal activity on Scarabaeidae insectsand growth inhibition on their larva.

Examples of microorganisms belonging to Bacillus popilliae that exhibitgrowth inhibitory or insecticidal activity against larva of Scarabaeidaeinsects include the bacterial species of Bacillus popilliae Semadara:FERM BP-8068, Bacillus popilliae var. popilliae Mame: FERM BP-8069,Bacillus popilliae var. popilliae Hime: FERM P-17660, Bacillus popilliaevar. popilliae Sakura: FERM P-17662, Bacillus popilliae Dutky: ATCC No.14706, and Bacillus popilliae subsp. melolonthae. Furthermore, Bacilluspopilliae Semadara was deposited at the National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology(currently the International Patent Organism Depository, NationalInstitute of Advanced Industrial Science and Technology) on May 21, 1998under the accession no. FERM P-16818, was transferred to internationaldeposition based on the Budapest Treaty on Jun. 10, 2002, and assignedthe accession no. FERM BP-8068. In addition, Bacillus popilliae var.popilliae Mame was deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology (currentlythe International Patent Organism Depository, National Institute ofAdvanced Industrial Science and Technology) on Nov. 25, 1999 under theaccession no. FERM P-17661, was transferred to international depositionbased on the Budapest Treaty on Jun. 10, 2002, and assigned theaccession no. FERM BP-8069.

On the other hand, examples of Scarabaeidae insects that can becontrolled include Anomala cuprea, Blitopertha orientalis, Popilliajaponica, Phyllopertha diversa, Adoretus tenuimaculatus and Anomalarufocuprea.

Sporangia of Bacillus popilliae containing spores and parasporal bodiesproduced according to the production process of the present inventionmay be used as a control agent for Scarabaeidae insects directly in theform of a liquid in which they are suspended. Alternatively, thesporangia may be dried and sprayed in the form of a powder. In addition,the sporangia may be dried followed by spraying onto the soil as asuspension with water or buffer. However, said sporangia are normallyformulated with commonly used additives used in agricultural chemicalsusing ordinary microbial agricultural chemical production methods, andthen preferably applied in the form of a control agent for Scarabaeidaeinsects. In addition, the sporangia of Bacillus popilliae containingspores and parasporal bodies obtained according to the productionprocess of the present invention can also be used by mixing with othermicrobial preparations. Although there are no particular restrictions onthe content ratio of sporangia of Bacillus popilliae containing sporesand parasporal bodies contained in the aforementioned control agentprovided it is within a range that demonstrates control effects onScarabaeidae insects, in the case of, for example, a water-dispersiblepowder or emulsion at the time of application, it is preferablyformulated so as to contain 1×10⁹ to 1×10¹³ sporangia per 1 liter ofcontrol agent, while in the case of a powder or granules, it ispreferably formulated so as to contain 1×10⁸ to 1×10¹² sporangia per 1gram of control agent.

The method for applying the control agent of the present invention issuitably selected according to the preparation form, target crop and soforth, examples of which include ground-level liquid spraying,ground-level solid spraying, aerial liquid spraying, aerial solidspraying, indoor application, soil mixing and soil perfusion. Inaddition, the control agent of the present invention can also be appliedby mixing with other chemicals such as insecticides, nematocides,miticides, herbicides, bactericides, plant growth regulators,fertilizers and soil improvers (such as peat, humus and polyvinylalcohol-based materials), or can be applied alternately orsimultaneously with other chemicals without being mixed with them.

Although the applied amount of the aforementioned control agent cannotbe specified unconditionally since it varies according to the type ofScarabaeidae insect, type of applied plant, preparation form and soforth, in the case of ground-level spraying, for example, the appliedamount of sporangia of Bacillus popilliae containing spores andparasporal bodies of the present invention is 10¹⁰-10¹⁵ sporangia/are,and preferably 10¹¹-10¹⁴ sporangia/are.

EXAMPLES

The following provides a more detailed explanation of the presentinvention through its examples and test examples.

Reference Example 1

The free amino acid contents of the peptone, yeast extract andlactoalbumin hydrolyzate added to the liquid media of each example weremeasured by the post column method described below usingorthophthalaldehyde (OPA).

(1) Sample Preparation

Mixed amino acid standard H (Wako Pure Chemical Industries, containing2.5 mmol/l of each amino acid) used as the reference sample was dilutedfive-fold with hydrochloric acid having a concentration of 0.02 mol/land filtered with a filter having a pore size of 0.2 μm to prepare thereference sample solution.

Measurement samples were prepared by preparing 1.0% by weight solutionsof Polypeptone S (Nippon Pharmaceutical) or Tryptone (Difco) for thepeptone, 1.0% by weight solutions of yeast extract manufactured by Oxoidor Difco for the yeast extract, and 1.0% by weight solutions oflactoalbumin hydrolyzate (Wako Pure Chemical Industries), followed bydiluting these solutions two-fold with 10% by weight aqueoustrichloroacetic acid solution, stirring well and centrifuging to removeany insoluble precipitates. Subsequently, the supernatant was filteredwith a filter having a pore size of 0.2 μm to prepare the measurementsample solutions.

(2) Analysis

10 μl aliquots of the reference sample solution and measurement samplesolutions were injected into a high-performance liquid chromatograph toanalyze the amino acids. Furthermore, amino acid analyses were carriedout using the Hitachi “LaChrom” Amino Acid Auto Analyzer. Furthermore,the compositions of the OPA labeling reaction solutions and eluates usedin the amino acid analyses are shown in Tables 2 and 3, respectively.TABLE 2 Composition of reaction solution for OPA labeling R1 R2 R3 Boricacid 21.6 g 21.6 g Sodium hydroxide 24.0 g 25% Brij-35 solution 4.0 ml4.0 ml o-phthalaldehyde/methanol 800 mg/ 10 ml 2-mercaptoethanol 2.0 ml5% sodium hypochlorite 150.0 μl solution Distilled Water RemainderRemainder Remainder Total volume 1,000 ml 1,000 ml 1,000 ml

TABLE 3 Eluate A B C Sodium citrate 2H₂O 8.14 g 26.67 g Sodium chloride7.07 g 54.35 g Citric acid H₂O 20.00 g 6.10 g Sodium hydroxide 8.0 gEthanol 110 ml Caprylic acid 0.1 ml 0.1 ml 0.1 ml Distilled WaterRemainder Remainder Remainder Total 1,000 ml 1,000 ml 1,000 ml

Reagents manufactured by Wako Pure Chemical Industries were used for allreagents, and amino acid analytical grade reagents were used for thesodium citrate 2H₂O, citric acid H₂O and caprylic acid, while guaranteedreagents were used for all others. The concentrations of proline andtotal amino acids contained in each of the measurement sample solutionswere calculated by converting from the peak areas obtained from thereference sample solution and each measurement sample solution, andthose concentrations are shown in Table 4. TABLE 4 Lacto- Peptonealbumin Polypeptone Yeast extract hydroly- S Tryptone Oxoid Difco zateProline 0.000 0.124 0.419 0.285 0.103 concentration (wt %) Total amino17.878 21.653 36.668 31.452 27.369 acid concentration (wt %)

Preparation Example 1

5 g of L-proline as added amino acid (guaranteed reagent, Wako PureChemical Industries), 5 g of peptone (“Polypeptone S”, NipponPharmaceutical), 5 g of yeast extract (Oxoid) and 5 g of trehalosedihydrate (guaranteed reagent, Wako Pure Chemical Industries were addedto a beaker containing 700 g of distilled water and mixed. Moreover,aqueous potassium hydroxide solution having a concentration of 5 mol/lwas added while stirring to adjust the pH to 7.6. Moreover, distilledwater was added to bring to a final weight of 850 g. This liquid mediumwas then transferred to a fermentation tank equipped with a pH electrode(B. E. Marubishi) and sterilized by autoclaving for 60 minutes at 121°C.

Next, 3 g of activated carbon powder (guaranteed reagent, Wako PureChemical Industries) were placed in a flask followed by the addition ofdistilled water to bring to a final weight of 100 g to prepare anactivated carbon dispersion. In addition, 1 g of antifoaming agent(Disfoam CA-123, NOF) was placed in a flask followed by the addition ofdistilled water to bring to a final weight of 50 g to prepare anantifoaming agent liquid. The activated carbon dispersion andantifoaming agent liquid were sterilized followed by their asepticaddition to the fermentation tank to prepare liquid medium (A).

Comparative Preparation Example 1

Liquid medium (B-1) was obtained in the same manner as PreparationExample 1 with the exception of not adding activated carbon powder inPreparation Example 1.

Comparative Preparation Example 2

Liquid medium (B-2) was obtained in the same manner as PreparationExample 1 with the exception of not adding L-proline in PreparationExample 1.

Comparative Preparation Example 3

Liquid medium (B-3) was obtained in the same manner as PreparationExample 1 with the exception of adding 5 g of L-alanine (guaranteedreagent, Wako Pure Chemical Industries) instead of the L-proline inPreparation Example 1. TABLE 5 Medium Name Medium A Medium B-1 MediumB-2 Medium B-3 Medium Added amino L-proline L-proline L-alanine compo-acid 5 g 5 g — 5 g nents Activated carbon 3 g — 3 g 3 g Peptone 5 g 5 g5 g 5 g Yeast extract 5 g 5 g 5 g 5 g Trehalose dihydrate 5 g 5 g 5 g 5g Antifoaming agent 1 g 1 g 1 g 1 g Distilled water Remainder RemainderRemainder Remainder Total Amount 1,000 g 1,000 g 1,000 g 1,000 g

Comparative Preparation Example 4

Liquid medium (B-4) was obtained by placing 80 g of distilled water in aflask, mixing in 0.5 g of peptone (“Tryptone”, Difco), 0.5 g of yeastextract (Oxoid) and 0.3 g of dipotassium hydrogenphosphate (guaranteedreagent, Wako Pure Chemical Industries), 0.1 g of glucose (guaranteedreagent, Wako Pure Chemical Industries) and 1.0 g of activated carbonpowder (guaranteed reagent, Wako Pure Chemical Industries), and addingdistilled water to bring to a final weight of 100 g followed bysterilizing in an autoclave for 20 minutes at 121° C. TABLE 6 MediumName B-4 Medium Activated carbon 1.0 g Components Tryptone 0.5 g Yeastextract 1.5 g Glucose 0.1 g Dipotassium hydrogenphosphate 0.3 gDistilled water Remainder Total Amount 100 g

Example 1

Sporangia of Bacillus popilliae Semadara (FERM BP-8068), Bacilluspopilliae var. popilliae Sakura (FERM P-17662) and Bacillus popilliaevar. popilliae Mame (FERM BP-8069) were cultured in advance by a knownmethod of solid culturing described in Japanese Unexamined PatentApplication, First Publication No. 2001-149066. Moreover, each of themicroorganisms were collected aseptically, and the number of sporangiacontaining spores and parasporal bodies in 1 ml of distilled water wasadjusted to 1×10⁹ sporangia by measuring by direct microscopicexamination to prepare sporangia liquids.

1 ml aliquots of the sporangia liquid of each strain were transferred toplastic tubes followed by heat treatment for 20 minutes at 70° C. usinga heating block. 1 ml of each sporangia liquid was inoculated intoliquid medium (A) in the aforementioned fermentation tank (B. E.Marubishi) followed by culturing for 7 days under controlled conditionsof aeration of 1 vvm, 30° C. and pH 7.6 while stirring the liquid mediumby rotating the stirrer provided with the fermentation tank at 150 rpm.

Following completion of culturing, the numbers of sporangia andmicrobial cells per unit volume in the liquid cultures were measured bydirect microscopic examination (Eclipse E600, Nikon, magnification:3800×) followed by calculation of the sporangia formation rate relativeto the number of microorganisms. The numbers of sporangia per 1 ml ofliquid culture and sporangia formation rates are shown in Tables 7through 9.

Comparative Examples 1-3

With the exception of using liquid media (B-1), (B-2) and (B-3),respectively, in place of liquid medium (A) in Example 1, culturing wascarried out in the same manner as Example 1, and the numbers ofsporangia containing spores and parasporal bodies as well as the numbersof microbial cells per unit volume in the liquid cultures were measuredfollowed by calculation of the sporangia formation rates. The numbers ofsporangia and sporangia formation rates per 1 ml of liquid culture areshown in Tables 7 through 9.

Comparative Example 4

With the exception of using liquid medium (B-4) in place of liquidmedium (A), and setting the rotation condition of stirrer to be 100 rpmin Example 1, culturing was carried out in the same manner as Example 1,and the number of sporangia containing spores and parasporal bodies aswell as the number of microbial cells per unit volume in the liquidculture were measured followed by calculation of the sporangia formationrate. The number of sporangia and sporangia formation rate per 1 ml ofliquid culture are shown in Tables 7 through 9. TABLE 7 Culture ofBacillus popilliae Semadara Proline Ratio of concentration proline toNo. of Sporangia Medium in liquid total amino sporangia formation Namemedium (wt %) acids (wt %) (sporangia/ml) rate (%) A 0.502 64.977  1.1 ×10⁸ 6.2 B-1 0.502 64.977 <1.0 × 10⁴ 0 B-2 0.002 0.768 <1.0 × 10⁴ 0 B-30.002 0.768 <1.0 × 10⁴ 0 B-4 0.007 1.049 <1.0 × 10⁴ 0

TABLE 8 Culture of Bacillus popilliae var. popilliae Sakura ProlineRatio of concentration proline to No. of Sporangia Medium in liquidtotal amino sporangia formation Name medium (wt %) acids (wt %)(sporangia/ml) rate (%) A 0.502 64.977  1.1 × 10⁸ 6.9 B-1 0.502 64.977<1.0 × 10⁴ 0 B-2 0.002 0.768 <1.0 × 10⁴ 0 B-3 0.002 0.768 <1.0 × 10⁴ 0B-4 0.007 1.049 <1.0 × 10⁴ 0

TABLE 9 Culture of Bacillus popilliae var. popilliae Mame Proline Ratioof concentration proline to No. of Sporangia Medium in liquid totalamino sporangia formation Name medium (wt %) acids (wt %) (sporangia/ml)rate (%) A 0.502 64.977  1.1 × 10⁸ 7.1 B-1 0.502 64.977 <1.0 × 10⁴ 0 B-20.002 0.768 <1.0 × 10⁴ 0 B-3 0.002 0.768 <1.0 × 10⁴ 0 B-4 0.007 1.049<1.0 × 10⁴ 0

Based on the results of Tables 7 through 9, sporangia are only obtainedin liquid media to which has been added adsorbent and proline, and saidsporangia were confirmed by microscopic observation to contain one sporeand one parasporal body.

Preparation Example 2

0.1 g of L-proline (guaranteed reagent, Wako Pure Chemical Industries),7.5 g of peptone (“Polypeptone S”, Nippon Pharmaceutical), 7.5 g ofyeast extract (Oxoid), 5 g of lactoalbumin hydrolyzate (guaranteedreagent, Wako Pure Chemical Industries) and 5 g of trehalose dihydrate(guaranteed reagent, Wako Pure Chemical Industries) were added to abeaker containing 700 g of distilled water and mixed. After adjustingthe pH to 7.6 by adding aqueous potassium hydroxide solution having aconcentration of 5 mol/l while stirring, distilled water was added tobring to a final weight of 850 g. This was then transferred to afermentation tank equipped with a pH electrode (B. E. Marubishi) andsterilized by autoclaving for 60 minutes at 121° C.

Next, 3 g of activated carbon powder (guaranteed reagent, Wako PureChemical Industries) were added to a flask followed by the addition ofdistilled water to bring to a final weight of 100 g to prepare anactivated carbon dispersion. In addition, 1 g of antifoaming agent(Disfoam CA-123, NOF) was added to a flask followed by the addition ofdistilled water to bring to a final weight of 50 g to prepare anantifoaming agent liquid. The activated carbon dispersion andantifoaming agent liquid were sterilized followed by their asepticaddition to each fermentation tank to obtain medium (C-1).

Preparation Examples 3 and 4

Liquid media (C-2) and (C-3) were respectively prepared in the samemanner as Preparation Example 2 with the exception of changing theamount of L-proline added to 0.2 g and 0.5 g, respectively, inPreparation Example 2.

Comparative Preparation Example 5

Liquid medium (D-1) was prepared in the same manner as PreparationExample 2 with the exception of not adding L-proline in PreparationExample 2.

Comparative Preparation Example 6

Liquid medium (D-2) was prepared in the same manner as PreparationExample 2 with the exception of changing the amount of L-proline addedto 0.8 g in Preparation Example 2. TABLE 10 Medium Name D-1 C-1 C-2 C-3D-2 Medium L-proline — 0.1 g 0.2 g 0.5 g 0.8 g compo- Activated carbon 3g 3 g 3 g 3 g 3 g nents Peptone 7.5 g 7.5 g 7.5 g 7.5 g 7.5 g Yeastextract 7.5 g 7.5 g 7.5 g 7.5 g 7.5 g Lactoalbumin hydrolyzate 5 g 5 g 5g 5 g 5 g Trehalose dihydrate 5 g 5 g 5 g 5 g 5 g Antifoaming agent 1 g1 g 1 g 1 g 1 g Distilled water Remainder Remainder Remainder RemainderRemainder Total Amount 1,000 g 1,000 g 1,000 g 1,000 g 1,000 g

Examples 2-4 and Comparative Examples 5-6

Sporangia of Bacillus popilliae Semadara (FERM BP-8068) were cultured inadvance by a known method of solid culturing described in JapaneseUnexamined Patent Application, First Publication No. 2001-149066.Moreover, the microorganisms were collected aseptically, and the numberof sporangia containing spores and parasporal bodies in 1 ml ofdistilled water was adjusted to 1×10⁹ sporangia by measuring by directmicroscopic examination to prepare a sporangia liquid.

1 ml aliquots of the sporangia liquid were transferred to plastic tubesfollowed by heat treatment for 20 minutes at 70° C. using a heatingblock. 1 ml each of sporangia liquid was inoculated into liquid media(C-1) through (C-3) and liquid media (D-1) through (D-2) followed byculturing for 7 days under the same conditions as Example 1.

Following completion of culturing, the numbers of sporangia andmicrobial cells per unit volume in the liquid cultures were measured bydirect microscopic examination followed by calculation of the sporangiaformation rates. The numbers of sporangia per 1 ml of liquid culture andsporangia formation rates are shown in Table 11. TABLE 11 Ratio ofL-proline L-proline content in to total No. of liquid amino microbialNo. of Sporangia Medium culture acids cells sporangia formation Name (wt%) (wt %) (cells/ml) (sporangia/ml) rate (%) D-1 0.004 0.670 5.6 × 10⁸<1.0 × 10⁴  0 C-1 0.104 16.048 1.5 × 10⁹ 8.2 × 10⁷ 5.5 C-2 0.204 27.3021.1 × 10⁹ 1.6 × 10⁸ 10.6 C-3 0.504 48.154 1.8 × 10⁹ 1.8 × 10⁸ 10.0 D-20.804 59.710 3.5 × 10⁸ <1.7 × 10⁷  0

One spore and one parasporal body were contained in the sporangiaobtained in Examples 2-4 using media (C-1) through (C-3). In addition,the relationship between proline concentration in the liquid medium andsporangia formation rate is shown in FIG. 2 based on results shown inTable 11. It can be seen from FIG. 2 that the optimum prolineconcentration range is within the range of 0.1-0.7% by weight.

Preparation Example 5

5 g of L-proline (guaranteed reagent, Wako Pure Chemical Industries), 1g of sodium pyruvate (guaranteed reagent, Wako Pure ChemicalIndustries), 7.5 g of peptone (“Polypeptone S”, Nippon Pharmaceutical),7.5 g of yeast extract (Oxoid), 5 g of lactoalbumin hydrolyzate (WakoPure Chemical Industries) and 5 g of trehalose dehydrate (guaranteedreagent, Wako Pure Chemical Industries) were added to a beakercontaining 700 g of distilled water and mixed. After adjusting the pH to7.6 by adding aqueous sodium hydroxide solution having a concentrationof 4 mol/l while stirring, distilled water was added to bring to a finalweight of 850 g. This was then transferred to a fermentation tankequipped with a pH electrode (B. E. Marubishi) and sterilized byautoclaving for 50 minutes at 121° C.

Next, 2.5 g of activated carbon powder (guaranteed reagent, Wako PureChemical Industries) were placed in a flask followed by the addition ofdistilled water to bring to a final weight of 100 g to prepare anactivated carbon dispersion. In addition, 1 g of antifoaming agent(Disfoam CA-123, NOF) was placed in a flask followed by the addition ofdistilled water to bring to a final weight of 50 g to prepare anantifoaming agent liquid. The activated carbon dispersion andantifoaming agent liquid were sterilized followed by their asepticaddition to the fermentation tank to prepare liquid medium (E-1).

Preparation Example 6

With the exception of using 2.5 g of sodium pyruvate added inPreparation Example 6, liquid medium (E-2) was obtained in the samemanner as Preparation Example 6.

Preparation Example 7

Liquid medium (F) was obtained in the same manner as Preparation Example6 with the exception of not adding L-proline in Preparation Example 6.TABLE 12 Medium Name E-1 E-2 F Medium L-proline 5 g 5 g — compo- Sodiumpyruvate 1 g 2.5 g 1 g nents Activated carbon 2.5 g 2.5 g 2.5 g Peptone7.5 g 7.5 g 7.5 g Yeast extract 7.5 g 7.5 g 7.5 g Lactoalbumin 5 g 5 g 5g hydrolyzate Trehalose dihydrate 5 g 5 g 5 g Antifoaming agent 1 g 1 g1 g Distilled water Remainder Remainder Remainder Total Amount 1,000 g1,000 g 1,000 g

Examples 5-6 and Comparative Example 7

Using Bacillus popilliae Semadara for the inoculating microorganisms inthe same manner as Example 2, 1 ml aliquots were aseptically inoculatedinto liquidmedia (E-1) through (E-2) and liquid medium (F), after whichculturing was started in the aforementioned fermentation tank (B. E.Marubishi). The culturing conditions consisted of a temperature of 29°C., aeration of 0.5 vvm and rotating the stirrer provided with thefermentation tank at 150 rpm, and during culturing, the pH wascontrolled to pH 7.6 with aqueous sodium hydroxide solution having aconcentration of 4 mol/l and sulfuric acid having a concentration of 4mol/l.

Culturing was carried out for 5 days, and the numbers of sporangia andmicrobial cells per unit volume in the liquid cultures were measured bydirect microscopic examination followed by calculation of the sporangiaformation rates. Those results are shown in Table 13. TABLE 13 L-prolineRatio of concen- L-proline tration in to total No. of liquid aminomicrobial No. of Sporangia Medium culture acids cells sporangiaformation Name (wt %) (wt %) (cells/ml) (sporangia/ml) rate (%) E-10.504 48.154 1.4 × 10⁹ 2.0 × 10⁸ 14.3 E-2 0.504 48.154 1.6 × 10⁹ 4.8 ×10⁸ 29.3 F 0.004 0.680 1.0 × 10⁹ <1.0 × 10⁴  0

One spore and one parasporal body were contained in the sporangiaobtained in Examples 5 and 6 using media (E-1) and (E-2). In addition,as is clear from the results shown in Table 13, the number of sporangiacontaining spores and parasporal bodies per unit volume of medium wasable to be further increased by adding sodium pyruvate and controllingthe pH.

Biological Test Example 1

A test was conducted on the growth inhibitory effects on larva ofScarabaeidae insects of sporangia obtained by the production process ofthe present invention.

Sporangia of Bacillus popilliae Semadara acquired in medium using liquidmedium (A) of Example 1 were suspended in distilled water to 2×10⁸sporangia/ml to prepare suspension (I). Moreover, a suspensioncontaining said sporangia was treated with a French press to separateand remove the spores and parasporal bodies from the sporangia. Theseparated spores were suspended in distilled water to 2×10⁸ spores/ml toprepare suspension (II). In addition, the separated parasporal bodieswere suspended in distilled water to 2×10⁸ parasporal bodies/ml toprepare suspension (III).

Eighty plastic cups were prepared having a diameter of 6 cm and filledwith about 20 g each of leaf mold.

-   (i) Suspension (I) containing sporangia containing spores and    parasporal bodies was sprayed onto 20 plastic cups so that the    number of sporangia was 2×10⁸ sporangia/cup.-   (ii) Suspension (II) containing spores only was sprayed onto 20    plastic cups so that the number of spores was 2×10⁸ spores/cup.-   (iii) Suspension (III) containing parasporal bodies only was sprayed    onto 20 plastic cups so that the number of parasporal bodies was    2×10⁸ parasporal bodies/cup.-   (iv) Nothing was sprayed onto the remaining 20 cups and these cups    were used as a control test.

One second instar larva each of Anomala cuprea was placed in each cupand bred for 30 days in an incubator at 25° C. followed by measuring themortality rates and average body weight increase of the surviving larvaover time. The cumulative mortality rates are shown in Table 14, whilethe results for growth inhibitory effects are shown in FIG. 3. TABLE 14Cumulative Mortality Rates (%) Test Group Day 11 Day 23 Day 30 (i) 20 4045 (ii) 0 5 10 (iii) 15 20 25 (iv) Control 0 0 0

As is clear from Table 14 and FIG. 3, sporangium containing both sporesand parasporal bodies were confirmed to demonstrate superiorinsecticidal and larva growth inhibitory effects on the larva ofScarabaeidae insects as compared with the case of spores alone and thecase of parasporal bodies alone.

Biological Test Example 2

A test was conducted of the insecticidal activity on Scarabaeidaeinsects by sporangia obtained according to the production process(liquid culturing) of the present invention.

Approximately 20 g of leaf mold were placed in 60 plastic cups having adiameter of 6 cm, and a sporangia liquid containing sporangia of (i) or(ii) below was sprayed onto 20 cups each so that the number of saidsporangia was 1×10⁹ sporangia/cup.

However, the sprayed sporangia liquids used in this test consisted of(i) sporangia of Bacillus popilliae Semadara containing spores andparasporal bodies acquired by culturing using liquid medium (A) ofExample 1, and (ii) sporangia of Bacillus popilliae var. popilliae Mameacquired by culturing in medium using liquid medium (A) of Example 1.

In addition, nothing was sprayed onto the remaining 20 cups and thesecups were used as a control test. One second instar larva each ofAnomala cuprea was placed in each cup and bred for 40 days in anincubator at 25° C. followed by investigating the number of insects thatdied over time to determine the cumulative mortality rates (%). Thoseresults are shown in Table 15. TABLE 15 Cumulative Mortality Rates (%)Test Group Day 10 Day 20 Day 30 Day 40 (i) 25 40 90 100 (ii) 15 40 75 80Control 0 0 0 0

Based on the results shown in Table 15, mortality rates of 80-100% wereobserved on day 40. Namely, sporangia containing spores and parasporalbodies of Bacillus popilliae were confirmed to have superiorinsecticidal and larval growth inhibitory effects on larva ofScarabaeidae insects.

Biological Test Example 3

A test was conducted of the insecticidal activity on Scarabaeidaeinsects by sporangia obtained according to the production process(liquid culturing) of the present invention. Sporangia of Bacilluspopilliae Semadara containing spores and parasporal bodies obtained byculturing in liquid medium (E-2) shown in Example 6 were suspended indistilled water to 1×10⁹ sporangia/ml to prepare a sporangia liquid.

Approximately 20 g of leaf mold each were placed in 40 plastic cupshaving a diameter of 6 cm. The sporangia liquid was sprayed onto 20 ofthe cups so that the number of sporangia was 1×10⁹ sporangia/cup.Sporangia liquid was not sprayed onto the remaining 20 cups and thesecups were used as a control test. One second instar larva each ofAnomala cuprea was placed in each cup and bred for 40 days in anincubator at 25° C. followed by investigating the number of insects thatdied over time to determine the cumulative mortality rates (%). Thoseresults are shown in Table 16. TABLE 16 Cumulative Mortality Rates (%)Test Group Day 10 Day 20 Day 30 Day 40 Control 0 0 0 0 Sporangia 10 4090 100 addition

Based on the results shown in Table 16, the resulting sporangiademonstrated insecticidal activity, with all of the larva having died byday 40. Namely, sporangia of Bacillus popilliae Semadara containingspores and parasporal bodies obtained in Example 6 were confirmed tohave superior insecticidal and larval growth inhibitory effects on larvaof Scarabaeidae insects.

1. A process for producing sporangia of Bacillus popilliae containingspores and parasporal bodies by culturing Bacillus popilliae in a liquidmedium containing an adsorbent and 0.1-0.7% by weight of proline.
 2. Aprocess for producing sporangia of Bacillus popilliae according to claim1, wherein the ratio of proline to total amino acids contained in theliquid medium is within the range of 10 to 65% by weight.
 3. A processfor producing sporangia of Bacillus popilliae according to claim 1,wherein the ratio of adsorbent contained in the liquid medium is 0.05-5%by weight.
 4. A process for producing sporangia of Bacillus popilliaeaccording to claim 1, wherein the liquid medium additionally containspyruvic acid.
 5. A process for producing sporangia of Bacillus popilliaeaccording to claim 4, wherein the ratio of pyruvic acid in the liquidmedium is within the range of 0.01 to 0.5% by weight.
 6. A process forproducing sporangia of Bacillus popilliae according to claim 1, 3 or 4,wherein the microorganism belonging to Bacillus popilliae is Bacilluspopilliae Semadara, Bacillus popilliae var. popilliae Mame, Bacilluspopilliae var. popilliae Hime or Bacillus popilliae var. popilliaeSakura.